Alzheimer's Disease (AD) is the most prevalent form of dementia that strikes 5 million Americans currently. Without effective prevention or therapeutics, the number of AD patients 65 years or older is projected to reach 13.5 million in 2050, with care costs exceeding 1 trillion dollars per year. AD and several other devastating neurodegenerative disorders are collectively known as tauopathies in which the tau protein becomes pathologically hyperphosphorylated and aggregates into the neurofibrillary tangles (NFT) in neuron cells. The resultant neuronal death and cognitive deficits lead to the progression of neurodegeneration of AD and other tauopathy patients. Discovering small chemicals that inhibit the formation of, or dissolve the pre-formed tangles is an attractive strategy that may mitigate or even revert the progression of AD. However, all of the purported tau aggregation inhibitors thus far have been discovered by targeting the non-pathological, unphosphorylated tau. Furthermore, animal studies suggested that the tangles may exert a protective function by sequestering the cytoplasmic hyperphosphorylated tau molecules. Accordingly, chemicals that liberate hyperphosphorylated tau from the tangles may actually be detrimental clinically. One of the reasons underlying these controversies is the lack of an efficient method that produces hyperphosphorylated tau protein for basic and translational studies. We have developed a system by which the tau protein phosphorylated by the GSK3 kinase can be effectively produced and purified in E. coli. Using the hyperphosphorylated recombinant tau protein as the target for drug discovery, pathophysiologically relevant therapeutic strategies can be established. This exploratory R21 project will achieve three major goals. Firstly, we will investigate the molecular details of how GSK3-phosphorylated tau forms the aggregate in vitro. Secondly, we will produce and compare tau isoforms resulting from different kinases that have been linked to Alzheimer's Disease. Thirdly, using the GSK3-phosphorylated tau, we will establish a drug screening assay and launch a pilot study that will lead to the discovery of small compounds that enhance or inhibit the propensity of hyperphosphorylated tau to form aggregates. Novelties and outlook. This R21 grant is an exploratory project in which we use a novel technology to produce hyperphosphorylated tau protein. For the first time, drug screen for tau aggregation will be conducted with a pathologically relevant isoform to identify both aggregation enhancers and inhibitors. By exploring these new territories, this project will lay a firm foundation for full-fledged basic and translational studies that eventually will lead to the discovery of novel therapeutic regimen and strategy. PUBLIC HEALTH RELEVANCE: Alzheimer's Disease strikes more than 5 million Americans currently and imposes tremendous economical, emotional, and societal burdens to this country. In this grant, we will use a novel ZAC technique to produce hyperphosphorylated tau protein that is intimately linked to the progression of Alzheimer's and several other neurodegenerative diseases. We will use this protein as a novel target to screen for compounds that modulate the propensity of hyperphosphorylated tau to aggregate, which eventually may lead to a specific therapeutic regimen that delays or stops the cognitive impairment of Alzheimer's Disease patients.